The novel circFKBP8/miR-432-5p/E2F7 cascade functions as a regulatory network in breast cancer

Background Circular RNAs (circRNAs) are capable of affecting breast cancer (BC) development. However, the role and underneath mechanism of circFKBP8 (also known as hsa_circ_0000915) in BC remain largely unknown. Methods Expression analyses were performed using quantitative real-time polymerase chain reaction (qRT-PCR), western blot, and immunohistochemistry (IHC) assays. Effects on cell functional phenotypes were determined by assessing cell proliferation, migratory capacity, invasion, and stemness in vitro. The relationship between microRNA (miR)-432-5p and circFKBP8 or E2F transcription factor 7 (E2F7) was examined by RNA pull-down, dual-luciferase reporter, and RNA immunoprecipitation (RIP) assays. Xenograft assays were used to identify the function of circFKBP8 in vivo. Results CircFKBP8 was presented at high levels in BC tissues and cells. High circFKBP8 expression was associated with worse overall survival in BC patients. CircFKBP8 suppression inhibited BC cell proliferation, migratory capacity, invasion and stemness in vitro. CircFKBP8 suppression blocked xenograft tumor growth in vivo. Mechanistically, circFKBP8 functioned as a miR-432-5p sponge to modulate E2F7 expression. CircFKBP8 modulated BC cell malignant behaviors by miR-432-5p, and miR-432-5p affected these cell phenotypes through E2F7. Conclusion Our observations prove that circFKBP8 promotes BC malignant phenotypes through the miR-432-5p/E2F7 cascade, offering a promising therapeutic and prognostic target for BC. Supplementary Information The online version contains supplementary material available at 10.1186/s41065-024-00331-1.


Introduction
Breast cancer (BC) is a serious malignant tumor with significant incidence and mortality among women worldwide [1,2].With the deterioration of lifestyle and ecological environment, the percentage of new cases of BC in China is as high as 3-4% annually [3].Therefore, elucidating the pathogenesis of BC, controlling the incidence of BC and improving the therapeutic effect of BC have become major problems to be solved.
Unlike linear RNA, circular RNAs (circRNAs) have a closed-loop structure and are a special class of noncoding RNA, so they are conserved and tissue-specific and not easily degraded by nucleic acid exonucleases [4,5].Studies have shown that circRNAs are involved in the occurrence and development processes of various tumors by acting as transcription regulators or sponges of microRNAs (miRNAs) [6].For example, circSEC24A was upregulated in pancreatic cancer (PC), and increased circSEC24A expression promoted PC progression by upregulating TGFBR2 via miR-606 [7].Circ_0001367 was reduced in glioma, and its upregulation could enhance LUZP1 expression to inhibit glioma progression by sponging miR-545-3p [8].Hsa_circ_0008434 was capable of promoting gastric cancer growth and metastasis via the miR-6838-5p/USP9X cascade [9].Many studies have demonstrated that circRNAs possess critical functions in BC progression.For instance, wan et al. found that circTFF1 regulated BC progression through sponging miR-338-3p and promoting FGFR1 expression [10].Zhong et al. revealed that circRASSF2 accelerated BC cell malignant behaviors through regulating the miR-1205/ HOXA1 cascade [11].Circ_0084927 was enriched in BC and facilitated BC progression via sponging miR-142-3p to modulate ERC1 expression [12].
According to a previous study, we found that many circRNAs were dysregulated in BC, of which circFKBP8 (also called hsa_circ_0000915 based on the circBase ID), a relatively unexplored circRNA in cancer biology, was significantly upregulated in BC [13], implying the implication of circFKBP8 in BC progression.Furthermore, the function and underlying mechanism of circFKBP8 in BC are unclear.Therefore, we sought to explore the precise action of circFKBP8 in BC.

Tissue sample
70 pairs of BC tissue specimens and adjacent non-cancer tissue specimens were obtained from BC cases with the written informed consent at Yichun People's Hospital & The Affiliated Yichun Hospital of Nanchang University.All the patients enrolled in this study had not anti-cancer therapy before operation, and their clinicopathological features were presented in

Quantitative real-time polymerase chain reaction (qRT-PCR)
Total RNA was isolated in Trizol reagent (Invitrogen).The concentration of RNA was determined by Nano-Drop2000 (Thermo Fisher Scientific, Waltham, MA, USA).Total RNA was converted into cDNA under use of PrimeScript RT reagent kit (Exiqon, Aarhus, Denmark) with random primers and miRNA-specific primers (miR-432-5p: 5 The qPCR analysis was performed by SYBR Green I (Takara, Dalian, China) and specific primers.The relative RNA expression was measured by the 2 −∆∆Ct method.Primers were shown in Table 2. GAPDH (for circRNA and mRNA) and U6 (for miRNA) were used as the housekeeping genes.

RNase R treatment
Total RNA was obtained from BC cells.2.5 µg of RNA was cultured with 4 U of RNase R (20 U/µL, BioVision, Milpitas, CA, USA) at 37℃ for 0.5 h.Then, circFKBP8 and GAPDH levels were detected by qRT-PCR.

CircRNA localization assay
Total RNA was isolated from the nucleus and cytoplasm of BC cells using PARIS™ Kit (Invitrogen), according to the manufacturer's instructions.Then, cir-cFKBP8 amount was analyzed via qRT-PCR.U6 and GAPDH were used as nuclear and cytoplasmic controls, respectively.

CCK8 assay
2 × 10 3 BC cells were cultured in 96-well plates for 48 h.After that, 10 µL CCK8 reagent was added into cells for additional 2 h.Then, the absorbance (450 nm) value was detected using a microplate reader (Thermo Fisher Scientific).

5-Ethynyl-29-deoxyuridine (EDU) assay
BeyoClick™ EdU-647 kit (Beyotime, shanghai, china) was used to detect cell proliferation ability.In brief, BC cells grown in 96-well plates were incubated with 25 µM EDU solution.After 4 h, cells were treated with paraformaldehyde and Triton X-100 (Beyotime).Subsequently, cells were incubated with 100 µL EdU detection solution for 30 min, and cell nuclei were stained with 300 µL diamidine phenylindole (DAPI; Beyotime) for 20 min.Then, pictures were taken under the fluorescence microscope and the EDU-stained cells were scored relative to total cells.

Colony formation assay
Approximately 500 BC cells were seeded into 6-well plates and incubated for 2 weeks.Then, cells were fastened with methanol (Sigma-Aldrich) for 10 min and stained with crystal violet (Sigma-Aldrich) for 15 min.The number of colonies was counted by Image J software (NIH, Bethesda, MD, USA).

Transwell assay
BC cells in serum-free DMEM medium were plated into the upper transwell chamber (Corning Inc., Corning, NY, USA) precoated with (invasion assay) or without (migration assay) matrigel (Corning Inc.), and lower chamber was added with DMEM medium plus 20% FBS.After 24 h, the migrated and invaded cells were fixed and stained by 0.1% crystal violet (Sigma-Aldrich).Pictures of cells were photographed under a microscope.

RNA immunoprecipitation (RIP) assay
RIP assay was conducted by the RIP Kit (Geneseed, guangzhou, China).In brief, BC cell lysates were mixed with the magnetic beads labeled with anti-Ago2 (ab156780, Abcam) or anti-IgG (ab109489, Abcam) antibody for 12 h.After beads were digested by proteinase K, the enrichment levels of miR-432-5p and circFKBP8 were evaluated by qRT-PCR.

Statistical analysis
The results were expressed as mean ± standard deviation (SD).All assays were independently performed with three times.Graphpad Prism 8.0 software was used to analyze data.Two group differences were analyzed by Student's t-test or Wilcoxon signed-rank test.The multiple groups were compared by one-way analysis of variance with Kruskal-Wallis tests.P < 0.05 was defined as statistical significance.

CircFKBP8 was significantly upregulated in BC tissues and cells
First, we found that circFKBP8 expression was significantly upregulated in BC tissues compared with adjacent non-cancer tissues (Fig. 1A), and circFKBP8 expression was notably increased in BC patients with distant metastasis compared with no metastasis (Fig. 1B).Survival analysis presented that high circFKBP8 expression in BC patients was significantly associated with a poor overall survival (OS) (Fig. 1C).CircFKBP8 expression was strikingly increased in MDA-MB-231, MDA-MB-453, BT549 and MDA-MB-468 BC cells compared to normal MCF10A cells (Fig. 1D).Because of the more significant upregulation of circFKBP8 in MDA-MB-231 and MDA-MB-468 BC cells (P < 0.001), we used the two cell lines in the subsequent experiments.RNase R treatment assay demonstrated that circFKBP8 was more stable than the linear GAPDH mRNA when treatment with RNase R (Fig. 1E).

Knockdown of circFKBP8 inhibited BC cell proliferation, migration, invasion, and stemness
To explore the unknown functions of circFKBP8 in BC, we constructed circFKBP8 knockdown cells using three shRNAs targeting circFKBP8 (sh-circFKBP8#1, #2, and #3).According to the qRT-PCR results, three shR-NAs could significantly reduce circFKBP8 expression, and sh-circFKBP8#1 had the highest knockdown efficiency (Fig. 2A).CCK8 assay showed that cell viability was inhibited in sh-circFKBP8#1 and sh-circFKBP8#2 groups relative to the sh-NC group (Fig. 2B).EDU and colony formation assays were used to detect BC cell proliferation.The results demonstrated that circFKBP8 knockdown reduced the percent of EdU-positive cells (Fig. 2C) and the number of generated colonies (Fig. 2D) in MDA-MB-231 and MDA-MB-468 cells.We also performed transwell assay, and found that inhibition of cir-cFKBP8 greatly suppressed cell migration and invasion abilities (Fig. 2E-F).In addition, sphere formation assay was used to detect BC cell stemness.The results showed that depletion of circFKBP8 repressed MDA-MB-231 and MDA-MB-468 cell stemness ability (Fig. 2G).We also detected the protein levels of proliferation-related factor KI-67, invasion-related MMP2, and stemnessrelated protein Nanogin BC cells, and found that silencing of circFKBP8 significantly reduced the expression of KI-67, MMP2 and Nanog proteins (Fig. 2H).These data revealed that circFKBP8 knockdown inhibited BC cell malignant behaviors in vitro.

CircFKBP8 targeted mir-432-5p in BC
To explore the underneath mechanism of circFKBP8, we analyzed the location of circFKBP8 in BC cells.As shown in Fig. 3A, circFKBP8 was mainly located in the cytoplasm of MDA-MB-231 and MDA-MB-468 cells.

CircFKBP8 promoted BC tumor growth in vivo
Xenograft assays were used to study the role of circFKBP8 on tumor growth in vivo.After knockdown of circFKBP8, tumor volume and weight were remarkably reduced (Fig. 8A-B).The expression of E2F7, MMP2 and Nanog was decreased in tumor tissues with sh-circFKBP8#1 transduction (Fig. 8C).Moreover, the tumors with sh-circFKBP8#1 transduction had fewer KI-67-positive cells (Fig. 8C).After sh-circFKBP8#1 transduction, the expression of circFKBP8 and E2F7 mRNA was remarkably decreased, but miR-432-5p expression was increased in tumor tissues (Fig. 8D).In addition, circFKBP8 silencing significantly inhibited the protein levels of E2F7, KI-67, MMP2 and Nanog in tumor tissues (Fig. 8E).These data demonstrated that circFKBP8 functioned as an oncogenic circRNA to promote BC tumor growth.

Discussion
BC is the most prevalent malignancy [14] and the second leading cause of cancer death in women worldwide [15].Therefore, it is critical to identify promising therapeutic targets for BC treatment.Increasing evidence suggests that circRNAs are involved in cancer progression [16][17][18][19].For example, circ_0008035 accelerated proliferation and migration of gastric cancer cells [20].CircCYP24A1 impeded tumorigenesis, cell migration and invasion of renal cell carcinoma [21].Also, circRNA_002178 knockdown was shown to suppress malignant phenotypes of BC cells [22].In our study, circFKBP8 was upregulated in BC tissues and cells.CircFKBP8 inhibition suppressed BC cell proliferation, migration, invasion, and stemness.Thus, circFKBP8 could promote malignant behaviors of BC cells in vitro.In addition, silencing of circFKBP8 limited tumor growth in vivo.These results together demonstrated that circFKBP8 acted as an oncogenic factor in BC.
CircRNAs are reported as important regulators in various cancers by acting as sponges of certain miRNAs to mitigate the inhibition of miRNAs on their downstream genes [23][24][25].MiR-432-5p repressed colorectal cancer cell growth and invasion through regulating CXCL5 expression [26].MiR-432-5p inhibited BC cell progression by targeting SLBP [27].In our study, we demonstrated that miR-432-5p was significantly reduced in BC.MiR-432-5p inhibition restored the suppression effects of circFKBP8 knockdown on BC cell malignant behaviors, indicating that miR-432-5p was a tumor suppressor in BC.The oncogenic function of circFKBP8 may be, at least in part, achieved by miR-432-5p reduction.
E2F7 is a known transcriptional factor and acts as a tumor promoter in various cancer [28,29].Overexpression of E2F7 promoted BC cell proliferation [30].Consistent with the previous study, our data showed that knockdown of E2F7 suppressed cell malignant progression in BC.Furthermore, E2F7 was confirmed as a downstream target of miR-432-5p, and overexpression of E2F7 could rescue anti-tumor activity of miR-432-5p overexpression in BC cells.Thus, E2F7 promoted BC cell progression, and miR-432-5p functioned as a tumor inhibitor in BC via targeting E2F7.The circRNA/miRNA/ mRNA axis has been researched in BC progression, such as circ_0103552/miR-515-5p/CYR61 and circ_000554/ miR-182/ZFP36 networks [31,32].Herein, we found that To sum up, our in vitro and in vivo data suggested that circFKBP8 functioned as an oncogenic factor in BC by sponging miR-432-5p to upregulate E2F7 expression.Our findings suggest that circFKBP8 could serve as a novel therapeutic target for BC treatment.

Fig. 1
Fig. 1 CircFKBP8 was highly expressed in BC. (A) CircFKBP8 expression was detected by qRT-PCR in BC tissues (n = 70) and adjacent non-cancer tissues (NC) (n = 70); (B) circFKBP8 expression in BC patients with no metastasis and distant metastasis; (C) The prognostic value of circFKBP8 by survival curve analysis; (D) circFKBP8 expression in BC cells (MDA-MB-231, MDA-MB-453, MDA-MB-468, and BT549) and human normal breast cells MCF-10 A; (E) The expression of circFKBP8 and GAPDH was detected after treatment with or without RNase R in BC cells.** P < 0.01, *** P < 0.001

Fig. 2
Fig. 2 CircFKBP8 inhibition suppressed BC cell proliferation, migration, invasion, and stemness.(B) The expression of circFKBP8 was detected after transfected with sh-NC, sh-circFKBP8#1, sh-circFKBP8#2, and sh-circFKBP8#3 in BC cells; (B-D) CCK-8 assay, EDU assay and colony formation assay were used to detect cell proliferation; (E-F) Transwell migration and invasion assays were performed to assess cell migration and invasion ability; (G) Sphere formation assay was used to evaluate cell stemness; (H) The protein levels of KI-67, MMP2 and Nanog in BC cells were detected by western blot assays.** P < 0.01, *** P < 0.001

Fig. 5
Fig. 5 E2F7 was a direct target of miR-432-5p.(A) The binding sites was predicted by starbase between miR-432-5p and E2F7; (B) Luciferase activity was detected in dual-luciferase assay; (C) The protein expression of E2F7 was detected in different groups; (D) UALCAN was used to analyze E2F7 expression in BC tissues in TCGA database; (E-G) The mRNA (E) and protein (F-G) levels of E2F7 in BC tissues were analyzed by qRT-PCR and western blot/IHC assays; (H) Western blot was used for protein analysis of E2F7 in BC cells; (I) After BC cells were transfected with sh-NC + inh-NC, sh-circFKBP8#1 + inh-NC, or sh-circFKBP8#1 + inh-miR-432-5p, the protein level of E2F7 was detected.** P < 0.01, *** P < 0.001

Fig. 6
Fig. 6 E2F7 promoted BC cell proliferation, migration, invasion, and stemness.MDA-MB-231 and MDA-MB-468 cells were transfected with si-NC or si-E2F7.(A) The protein level of E2F7 was detected after knockdown of E2F7 in BC cells; (B-G) CCK-8 assay, EDU assay, colony formation assay, transwell migration and invasion assay, and sphere formation assay were used to detect cell proliferation, migration and invasion ability and cell stemness; (H) Western blot assay was used to detect the protein levels of KI-67, MMP2 and Nanog in BC cells.*** P < 0.001

Fig. 8
Fig. 8 CircFKBP8 knockdown inhibited BC tumor growth in vivo.Xenograft model of sh-NC or sh-circFKBP8#1 group was established in mice.(A-B) Tumor volume and Tumor weight were detected in Xenograft assay; (C) The levels of KI-67, E2F7, MMP2 and Nanog was assessed by IHC staining in different groups; (D) The expression of circFKBP8, miR-432-5p and E2F7 was detected by qRT-PCR in tumors from different groups; (E) The protein levels of KI-67, E2F7, MMP2 and Nanog were evaluated by western blot assays in tumors form different groups.** P < 0.01, *** P < 0.001

Table
. The Ethics Committee of Yichun People's Hospital & The Affiliated Yichun Hospital of Nanchang University approved this study with approval No.20,230,218.

Table 1
The clinicopathologic features in breast cancer patients